A hammerhead ribozyme inhibits ADE1 gene expression in yeast

Area of Science:

• Molecular Biology
• Yeast Genetics
• Biochemistry

Background:

• Ribozymes (Rz) are RNA molecules with catalytic activity.
• Understanding in vivo Rz activity is essential for gene regulation studies.
• A visual readout system is needed to assess Rz function in living cells.

Purpose of the Study:

• To establish a model system in Saccharomyces cerevisiae to study factors affecting in vivo ribozyme activity.
• To investigate the impact of ribozyme gene construct (cis vs. trans) on gene expression inhibition.
• To develop a reliable method for detecting ribozyme-mediated cleavage products in vivo.

Main Methods:

• Utilized Saccharomyces cerevisiae as a model organism.
• Employed plasmid constructs for expressing ribozymes (Rz) in cis or trans relative to the ADE1 gene.
• Assessed ribozyme activity via visual Red phenotype and ADE1 mRNA level quantification.
• Combined RNA ligation and PCR to detect 3' cleavage fragments.

Main Results:

• Ribozyme-linked inhibition of ADE1 gene expression, indicated by a Red phenotype, was observed only when the Rz gene was 5' linked to ADE1.
• A diminution of ADE1 mRNA levels correlated with the Red phenotype under specific linkage conditions.
• The 3' cleavage fragment resulting from Rz activity was successfully detected.

Conclusions:

• The developed yeast system provides a robust platform for studying in vivo ribozyme activity.
• The 5' linkage of the ribozyme gene to the target gene is critical for effective in vivo gene expression inhibition.
• The RNA ligation-PCR technique is suitable for detecting ribozyme-mediated cleavage events in vivo.